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The present invention relates to human papillomavirus antigen formulations which show increased antigen stability and reduced antigen aggregation and precipitation. The present invention also relates to methods of preparing adjuvanted HPV vaccines using the human papillomavirus antigen formulations disclosed herein. The present invention also relates to adjuvanted human papillomavirus vaccines generated from these human papillomavirus antigen formulations.
Papillomavirus (PV) infections occur in a variety of animals, including humans, sheep, dogs, cats, rabbits, monkeys, snakes and cows. Papillomaviruses infect epithelial cells, generally inducing benign epithelial or fibroepithelial tumors at the site of infection. Papillomaviruses are species specific infective agents.
Papillomaviruses are classified into distinct groups based on the host that they infect. Human papillomaviruses (HPVs) are further classified into more than 70 types based on DNA hybridization studies. PV types appear to be type-specific immunogens in that a neutralizing immunity to infection by one type of papillomavirus does not confer immunity against another type of papillomavirus.
In humans, different HPV types cause distinct diseases. HPV types 1, 2, 3, 4, 7, 10 and 26-29 cause benign warts in both normal and immunocompromised individuals. HPV types 5, 8, 9, 12, 14, 15, 17, 19-25, 36 and 46-50 cause flat lesions in immunocompromised individuals. HPV types 6, 11,34, 39, 41-44 and 51-55 cause nonmalignant condylomata of the genital or respiratory mucosa. HPV types 16, 18, 31, 33, 35, 45, and 58 cause epithelial dysplasia of the genital mucosa and are associated with the majority of in situ and invasive carcinomas of the cervix, vagina, vulva and anal canal.
Papillomaviruses are small (50-60 nm), nonenveloped, icosahedral DNA viruses that encode for up to eight early and two late genes. The open reading frames (ORFs) of the virus genomes are designated E1 to E8 and L1 and L2, where xe2x80x9cExe2x80x9d denotes early and xe2x80x9cLxe2x80x9d denotes late. L1 and L2 code for virus capsid proteins. The early (E) genes are associated with functions such as viral replication, transcriptional regulation and cellular transformation.
The L1 protein is the major capsid protein and has a molecular weight of 55-60 kDa. L2 protein is a minor capsid protein which has a predicted molecular weight of 55-60 kDa and an apparent molecular weight of 75-100 kDa as determined by polyacrylamide gel electrophoresis. Immunological data suggest that most of the L2 protein is internal to the L1 protein within the viral capsomere. The L1 ORF is highly conserved among different papillomaviruses. The L2 proteins are less conserved among different papillomaviruses.
The L1 and L2 genes have been identified as good targets for immunoprophylaxis. Some of the early genes have also been demonstrated to be potential targets of vaccine development. Studies in the cottontail rabbit papillomavirus (CRPV) and bovine papillomavirus (BPV) systems have shown that immunizations with recombinant L1 and/or L2 proteins (produced in bacteria or by using vaccinia vectors) protected animals from viral infection. Expression of papillomavirus L1 genes in baculovirus expression systems or using vaccinia vectors resulted in the assembly of virus-like particles (VLP) which have been used to induce high-titer virus-neutralizing antibody responses that correlate with protection from viral challenge. Furthermore, the L1 and L2 genes have been used to generate vaccines for the prevention and treatment of papillomavirus infections in animals.
Virus-like particles containing HPV11 L1 protein have been expressed in both insect and mammalian cell systems. Expression of VLPs in yeast cells offers the advantages of being cost-effective and easily adapted to large-scale growth in fermenters. However, the HPV11 L1 protein is expressed at low levels in yeast cells. This was observed to be a result of truncation of the HPV11 L1 mRNA. In contrast, the HPV6 L1 gene is transcribed as full-length mRNA and is expressed to high levels. By modifying the HPV6 L1 DNA to encode the HPV11 L1 protein, it is possible to facilitate the transcription of full-length mRNA resulting in increased HPV11 L1 protein expression.
The L1 and L2 genes have been used to generate vaccines for the prevention and treatment of papillomavirus infections in animals. HPV type 16 L1 and L2 genes have been cloned into a vaccinia virus vector and infected CV-1 mammalian cells with the recombinant vector to produce virus-like particles (VLP).
Bacterially-derived recombinant bovine papillomavirus L1 and L2 have been generated. Neutralizing sera to the recombinant bacterial proteins cross-reacted with native virus at low levels, presumably due to differences in the conformations of the native and bacterially-derived proteins.
Recombinant baculoviruses expressing HPV16 L1 or HPV16 L2 ORFs have been used to infect insect SF9 cells and produce L1 and L2 proteins. Western blot analyses showed that the baculovirus-derived L1 and L2 proteins reacted with antibody to HPV16. The baculovirus derived L1 forms VLPs.
Jansen et al. (1995, Vaccine 13(16):1509-1514) use a running buffer comprising sodium chloride and Tween 80(copyright) during purification of L1 and L1+L2 VLPs from cottontail rabbit papillomavirus.
At present, purified recombinant HPV VLP formulations must be stored at high NaCl concentrations to prevent aggregation in solution. At low ionic strengths, HPV VLPs aggregate to the point of being precipitated out of solution. Based on these and other related observations, HPV bulk solutions have been stored frozen in the presence of high concentrations of NaCl (1.25-2.5 M). Highly aggregated samples of HPV 11 VLP manifest poor in vitro antigenicity as measured by RIA, EIA or BIA core assays. Therefore, a need exists to prepare an aqueous HPV VLP formulation which is stable at physiological salt conditions as well as under acceptable long term storage conditions. The present invention addresses and meets this need.
The present invention relates to human papillomavirus (HPV) antigen formulations which prevent antigen aggregation and increase antigen stability at physiological salt concentrations in the presence of a surfactant.
The present invention also relates to the generation of an adjuvanted HPV vaccine which is formed by mixing an HPV antigen formulation of the present invention with a biologically effective amount of an adjuvant to form an adjuvanted HPV vaccine.
The HPV antigen formulations and adjuvanted vaccines of the present invention include but are not solely limited to, as the antigen component, virus-like particles generated as a recombinant HPV subunit vaccine comprising either L1 or a combination of L1 and L2 proteins, from HPV types 6a, 6b, 11, 16 and 18. It is within the scope of this invention to stabilize monovalent forms of this recombinant vaccine as well as divalent forms (such as but in no way limited to recombinant HPV 11 L1, HPV 16 L1 and HPV 6a L1), and multivalent forms (such as but in no way limited to recombinant HPV 11 L1, HPV 6a L1, HPV 16 L1 and HPV 18 L1).
The present invention also relates to HPV antigen formulations which comprise a physiological salt concentration and a surfactant to provide increased stabilization of the vaccine component of the formulation at temperatures above 0xc2x0 C. The HPV formulations of the present invention should be amenable to prolonged storage for periods up to at least one month to about two years at about 2xc2x0 C. to about 8xc2x0 C.
An embodiment of the present invention relates to HPV antigen formulations wherein the formulation comprises a physiologically acceptable salt, including but not necessarily limited to sodium chloride, sodium sulfate, and ammonium sulfate. The purpose of inclusion of a salt in the formulation is to attain the desired ionic strength. Contributions to ionic strength may come from ions produced by the buffering compound, including but not limited to phoshate, citrate, acetate, succinate, Tris-HCl, MOPS, etc., as well as from the ions of non-buffering salts.
Another embodiment of the present invention relates to HPV antigen formulations wherein the formulation comprises a non-ionic surfactant, including but not necessarily limited to polyoxyethylene sorbitan fatty acid esters (Polysorbates) such as Polysorbate 80 (e.g., Tween 80(copyright)), Polysorbate 60 (e.g., Tween 60(copyright)) and Polysorbate 20 (e.g., Tween 20(copyright)), polyoxyethylene alkyl ethers (e.g., Brij 58(copyright), Brij 35(copyright)), as well as others including but not limited to Triton X-100(copyright), Triton X-114(copyright), NP40(copyright), Span 85 and the Pluronic series of non-ionic surfactants (e.g., Pluronic 121).
An additional emobodiment of the present invention relates to an HPV antigen formulation wherein the formulation comprises a non-ionic surfactant as disclosed above and present in a range up to about 0.2% w/v, the physiologically acceptable salt being sodium chloride at a concentration from about 10 mM to about 500 M, in the presence of a physiologically acceptable buffer.
Another emobodiment of the present invention relates to an HPV antigen formulation wherein the formulation comprises a non-ionic surfactant as disclosed above and present in a range up to about 0.2% w/v, the physiologically acceptable salt being sodium chloride at a concentration from about 50 mM to about 400 mM, in the presence of a physiologically acceptable buffer.
Yet another emobodiment of the present invention relates to an HPV antigen formulation wherein the formulation comprises a non-ionic surfactant as disclosed above and present in a range up to about 0.2% w/v, the physiologically acceptable salt being sodium chloride at a concentration from about 150 mM to about 300 mM, in the presence of a physiologically acceptable buffer.
Another embodiment of the present invention relates to a HPV antigen formulation wherein the physiologically acceptable salt is sodium chloride at a concentration from about 10 mM to about 500 mM, and the non-ionic surfactant, Polysorbate 80 (including but not limited to Tween 80(copyright)), is present in a range up to about 0.2% w/v, in the presence of a physiologically acceptable buffer.
A specific embodiment of the present invention relates to a HPV antigen formulation wherein the physiologically acceptable salt is sodium chloride at a concentration from about 10 mM to about 500 mM, and the non-ionic surfactant, Polysorbate 80 (including but not limited to Tween 80(copyright)), is present in a range from about 0.01% to about 0.1% w/v, in the presence of a physiologically acceptable buffer.
A preferred embodiment of the present invention relates to a HPV antigen formulation wherein sodium chloride is present in concentration from about 50 mM to about 400 mM, Polysorbate 80 (including but not limited to Tween 80(copyright)), is present in a percentage range in amounts from about 0.01% to about 0.1% w/v, in the presence of a physiologically acceptable buffer.
An especially preferred embodiment of the present invention relates to a HPV antigen formulation wherein sodium chloride is present in concentration from about 150 mM to about 300 mM, Polysorbate 80 (including but not limited to Tween 80(copyright)), is present in a percentage range in amounts from about 0.01% to about 0.1% w/v, in the presence of a physiologically acceptable buffer.
It will be known to one of skill in the art to provide the HPV antigen formulations of the present invention in a physiologically acceptable buffer, preferably but not necessarily limited to a formulation buffered by phosphate, citrate, acetate, succinate, Tris-HCl, or MOPS, preferably but not limited to a pH range from about pH 5.0 to 9.0, and especially within a pH range of about pH 6.0 to about 8.0.
The present invention also relates to methods of generating HPV vaccine formulations which involve using the HPV antigen formulation of the present invention. These improved methods of generating either an alum- or non-alum based HPV vaccine are described herein.
The present invention also relates to adjuvanted HPV vaccines wherein an biologically effective amount of an adjuvant is combined with a biologically effective amount of an antigen-containing formulation as disclosed herein.
The term xe2x80x9cPVxe2x80x9d as used herein is the abbreviation for xe2x80x9cpapillomavirus.xe2x80x9d
The term xe2x80x9cHPVxe2x80x9d as used herein is the abbreviation for xe2x80x9chuman papillomavirus.xe2x80x9d
The term xe2x80x9cVLPxe2x80x9d as used herein is the abbreviation for xe2x80x9cviral-like particle.xe2x80x9d
The term xe2x80x9cphysiologically acceptablexe2x80x9d as used herein means a buffer, an excipient or a salt wherein either the concentration or ionic strength is such that the formulation is biologically compatible with the immunized target host, such as a human.